Biomimetic Mineral Synthesis by Nanopatterned Supramolecular-Block Copolymer Templates

Supramolecular structures of matrix proteins in mineralizing tissues are known to direct the crystallization of inorganic materials. Here we demonstrate how such structures can be synthetically directed into predetermined patterns for which functionality is maintained. The study employs block copolymer lamellar patterns with alternating hydrophilic and hydrophobic regions to direct the assembly of amelogenin-derived peptide nanoribbons that template calcium phosphate nucleation by creating a low-energy interface. Results show that the patterned nanoribbons retain their β-sheet structure and function and direct the formation of filamentous and plate-shaped calcium phosphate with high fidelity, where the phase, amorphous or crystalline, depends on the choice of mineral precursor and the fidelity depends on peptide sequence. The common ability of supramolecular systems to assemble on surfaces with appropriate chemistry combined with the tendency of many templates to mineralize multiple inorganic materials implies this approach defines a general platform for bottom-up-patterning of hybrid organic–inorganic materials.

water and drying.The BCP samples were fabricated using established materials and methods described below.
BCP film preparation: Substrates were initially cleaned by oxygen plasma treatment (March Plasma CS1701F, 100 mTorr, 20 W, 60 s).The PS-r-PMMA-OH brush was then grafted to the substrate as described previously (4) to provide a "neutral" surface with balanced surface energies between PS and PMMA that helps ensure vertical orientation of the lamellar domains.Briefly, this is accomplished by spincoating the PS-r-PMMA-OH solution onto samples at 1500 rpm, baking the samples for 5 minutes at 252 °C in a nitrogen-enriched environment to promote the brush grafting to the substrate, and rinsing in PGMEA at 3000 rpm to remove ungrafted polymer brush.Polymer blend solutions were prepared by mixing different polymer solutions (all 2% by weight in PGMEA) in prescribed weight ratios to achieve the desired polymer mass ratios.Binary blends of diblock copolymers or ternary PS-b-PMMA/PS/PMMA blends were then spin-cast at 1500 rpm to achieve films with thicknesses ranging from 60 to 90 nm.Self-assembly was achieved by thermal or solvent vapor annealing (SVA).Samples with varying linewidths referred to in the main text were obtained from the ternary block copolymer/homopolymer blends by adjusting the blend composition or solvent vapor annealing protocol; the salient preparation details corresponding to each pattern designation used in the main text are provided in Table 1.BCP film annealing: Thermal annealing was performed by baking samples for 5 minutes at 252 °C in a nitrogen-enriched environment.Solvent vapor annealing was performed as described previously (5) by placing samples in a chamber with a controlled vapor pressure of either THF or acetone.Briefly, the annealing chamber consisted of a canister machined from a single aluminum block with a central pedestal to hold samples above an annular solvent reservoir.A Peltier plate is used to maintain a constant temperature of 21.0 ± 0.2 °C during annealing.Annealing is commenced by placing a sample on the pedestal, adding 4-5 mL of solvent to the reservoir, and covering the chamber with a 6.5 mm thick borosilicate glass window.Nitrogen purging at flow rates from 0 or 0.4 standard cubic centimeters per minute (SCCM) was used to control the maximum polymer film swelling, which was monitored in situ by measuring the film thickness at 20 s intervals during annealing using spectral reflectance in the visible range (Filmetrics F20-UV).Minimum polymer volume fractions (given by the ratio of initial to maximum solvent swollen film thickness) during solvent vapor annealing were in the range of 0.28 to 0.3 for THF, and 0.33 for acetone.After 1 hour, annealing was rapidly quenched by removing the glass cover, drying within ~1 s.After solvent vapor annealing, samples were baked for 30-60 s at 252 °C in air.

Substrates for mineralization
BCP samples without a peptide coating were used directly after cleaning.For peptide coated BCPs, 20-100 μL of freshly made 0.05 mg/mL peptide solution was drop-cast on a cleaned BCP surface and incubated at 37 ºC in a sealed chamber (Relative Humidity ~100%) for 1 hour following our previous protocol for HOPG.After incubation, solutions on HOPG were gently exchanged with 1 mM HCl (pH 3.1) first then H2O, three times each, to remove unbound protein/peptide without disassembly.
For mineralization without AFM (ToC figure) described in main text, the BCP samples were incubated in 0.05 mg/mL of the peptide solution for 1 hours at 37 ºC in sealed tube.The substrates were then immersed in 1 mM HCl (pH 3.1) first then H2O, three times each for 2 seconds, to remove unbound protein/peptide without disassembly.Subsequently, 1.5 mM CaCl2 and 14.9 mM KH2PO4 (pH 7.4) were independently prepared, mixed in a tube and the BCP substrates were incubated in the mixture for at least 20 minutes for nucleation and growth to take place.Following this, the samples were immersed in water to remove the excess mineral solution and dried in vacuum for AFM and TEM analysis.

Photoinduced Force Microscopy (PiFM)
BCP samples without a peptide coating were used directly after cleaning.For peptide coated BCPs, the substrates for mineralization described above were used for the PiFM after removing the excess water and allowing the wet peptide film to dry slowly in vacuum.All samples were characterized in air using a VistaScope PiFM (Molecular Vista Inc.) coupled to a Laser Tune QCL with a wave number resolution of 0.5 cm -1 and a tuning range from 800-1800 cm -1 .The microscope was operated in dynamic mode with HQ:NSC15/Cr-Au probes (MikroMasch).The data was processed in Surface Works software (Molecular Vista Inc.).

In situ Atomic Force Microscopy (AFM)
Silicon nitride cantilevers with Si tip (Bruker SNL-10, spring constant k: 0.12 N/m or 0.24 N/m) treated with UV/ozone for 15 minutes were used for all experiments.Peptide self-assembly and ACP nucleation were performed using Bruker MultiMode 8 AFM, operated in liquid using tapping mode at room temperature (25˚C).PILP experiments were performed on the Cypher VRS AFM (Asylum Research) with a Peltier heater/cooler stage to adjust sample temperature and operated in liquid.Gwyddion and ImageJ/Fiji software were used for offline processing of images, feature size measurements and counting nuclei.Detailed methodologies are available in a previous publication (1).
NR self-assembly: The bare BCP surface was first imaged in 10 mM HCl (pH 1.94) to ensure absence of contaminants, then 200 µL of freshly made 0.05 mg/mL peptide solution was injected into the AFM liquid cell and imaged immediately.
Measurements of peptide layer thickness: Line profiles (more than 10 for each type) with line width of 3 pixels were drawn perpendicular to each stripe, e.g., Figure 1b.Average thickness of peptide layers (NRs or monomers) on PS or PMMA is obtained from the difference of the average height of the stripe between coated and uncoated samples.To obtain height values of individual stripes, the boundary between PS and PMMA stripes was used as a baseline.The boundary appears as a valley in Figure 1b (white dashed line) or as the darkest regions in height images of Figure 1a.Some regions of the boundary remain uncoated with peptide when exposed to peptide solutions, therefore, suitable to obtain thickness from the peak value.
Nucleation and growth experiments: All BCP samples were characterized in pure water before flowing the mineral precursor solutions.The protocol and analyses for mineralization experiments at constant composition (supersaturation, σACP = 0.04) are identical to procedures used for amelogenin nanoribbon coated HOPG in a previous publication (1).Briefly, 1.5 mM CaCl2 and 14.9 mM KH2PO4 (pH 7.4) were independently prepared and filtered three times with a cellulose acetate filter (pore size of 0.1 μm) before immediate use.The filtered CaCl2 and KH2PO4 solutions were independently and continuously pumped at 37.25 μl/min and combined at the inlet of the AFM liquid cell using a custom T-junction.
Calculation of nucleation and growth rates: The nucleation and growth rates are extracted from AFM images using established methods (1).To determine nucleation rate specifically on PS or PMMA stripes shown in Table 2, the number of nuclei on PS and PMMA stripes were tracked and counted separately and normalized with the total area of their corresponding regions (PS or PMMA) in the entire image.

Sequence
Nucleation rate (J0 in nuclei µm PILP-based mineralization: All BCP substrates were first characterized in pure water then exposed to the PILP solution.In this method, the AFM Peltier cooler/heater stage temperature was set to 25 ˚C.After the sample temperature equilibrated, the cantilever holder was disassembled and the water on the sample was rapidly exchanged with the PILP solution twice without dehydration of the peptide film on the surface.The PILP solution was incubated on the substrate for 15 min, then the water on the cantilever holder was replaced with 25 µL of PILP solution to avoid dilution.Immediately after this step, the holder is rapidly brought in contact with the liquid on the BCP and close to the surface (to avoid drying at the edges due to necking/meniscus) and imaged.Experiments at 10 ˚C were performed similarly but by changing the stage temperature, which produced results identical to those shown in main text (Figure 3c).

Transmission Electron Microscopy (TEM)
Sample preparation: Immediately after mineralization, the substrate was rinsed with 200 µL of ultrapure water to quench the reaction and remove the mineral precursor solution and unbound particles.The substrate was then immediately dried with N2 gas.The mineral particles were extracted from the surface by using a pipette to drop-cast 5-10 µL of ethanol on the BCP surfaces and withdrawing the liquid after 30 seconds.The ethanol causes the BCP film to swell (characterized by AFM), which release the surfacebound mineral particles into the withdrawn solution.The ethanol and mineral mixture are then immediately drop-cast onto lacey carbon grids (Ted Pella, USA) and the solution is left to incubate on the grid for 5 min before removing the excess and allowing the grids to dry in ambient air before storage in vacuum until characterization.To verify whether there was any change in crystallinity during extraction, we prepared multiple samples, mineralized them for different durations of time, and verified the morphology by AFM and then by TEM, following a procedure developed and used previously for analysis of calcium phosphate phases nucleated on surfaces (1,6,7).
High-resolution TEM (HRTEM) and Selected Area Electron Diffraction (SAED): All grids were imaged with a field emission Titan ETEM 80-300 kV (Thermo Fisher Scientific) operated at 300 kV.Detailed characterization of the ACP and apatite particles is described elsewhere (1,6).Briefly, the FFT (Fast Fourier Transform) of HRTEM on several particles characterized as ACP lacked lattice fringes corresponding to crystalline calcium phosphate and SAED shows a broad diffraction band where d-spacing for crystalline calcium phosphate is expected (8).In contrast, for particles characterized as apatite in PILP experiments, the FFT of HRTEM images and SAED showed the lattice fringes and sharp bands at dspacings, respectively, for apatite.